A chromosomal locus required for copper resistance, competitive fitness, and cytochrome c biogenesis in Pseudomonas fluorescens

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 93; no. 14; pp. 7315 - 7320
• Main Authors: Yang, C.H. (University of California, Riverside, CA.), Azad, H.R, Cooksey, D.A
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 09.07.1996; National Acad Sciences; National Academy of Sciences
• Subjects: Amino Acid Sequence; BACTERIA; Bacteria - genetics; Base Sequence; Binding Sites; Chromosome Mapping; Chromosomes, Bacterial; CITOCROMO C; CLONACION; CLONAGE; COBRE; COMPETENCIA BIOLOGICA; COMPETITION BIOLOGIQUE; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; Conjugation, Genetic; Conserved Sequence; Copper; Copper - pharmacology; Cosmids; CUIVRE; CYTOCHROME C; Cytochrome c Group - biosynthesis; Cytochrome c Group - genetics; Cytochromes; Disulfides; DNA; DNA - chemistry; DNA Primers; DNA Transposable Elements; Drug Resistance, Microbial; Escherichia coli - genetics; GENE; GENES; Genetic Complementation Test; Genetic loci; Genomic Library; LOCI; LOCUS; Microbiology; Molecular Sequence Data; Mutagenesis, Insertional; Neurospora crassa - genetics; Open reading frames; Oxidases; Oxidoreductases - chemistry; OXIDORREDUCTASAS; OXYDOREDUCTASE; PROTEINAS; PROTEINE; PSEUDOMONAS FLUORESCENS; Pseudomonas fluorescens - drug effects; Pseudomonas fluorescens - genetics; Pseudomonas fluorescens - physiology; RESISTANCE AUX FACTEURS NUISIBLES; RESISTENCIA A AGENTES DANINOS; Restriction Mapping; SECUENCIA NUCLEOTIDICA; Sequence Homology, Amino Acid; SEQUENCE NUCLEOTIDIQUE; SINTESIS DE PROTEINAS; Soils; SOL POLLUE; SUELO CONTAMINADO; SULFATE DE CUIVRE; SULFATO DE COBRE; SYNTHESE PROTEIQUE; TIPOS DE SUELOS CULTIVABLES; TRANSCRIPCION; TRANSCRIPTION; Transcription, Genetic; TYPE DE SOL DE CULTURE
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.14.7315

A chromosomal locus required for copper resistance and competitive fitness was cloned from a strain of Pseudomonas fluorescens isolated from copper-contaminated agricultural soil. Sequence analysis of this locus revealed six open reading frames with homology to genes involved in cytochrome c biogenesis in other bacteria, helC, cycJ, cycK, tipB, cycL, and cycH, with the closest similarity being to the aeg-46.5(yej) region of the Escherichia coli chromosome. The proposed functions of these genes in other bacteria include the binding, transport, and coupling of heme to apocytochrome c in the periplasm of these Gram-negative bacteria. Putative heme-binding motifs were present in the predicted products of cycK and cycL, and TipB contained a putative disulfide oxidoreductase active site proposed to maintain the heme-binding site of the apocytochrome in a reduced state for ligation of heme. Tn3-gus mutagenesis showed that expression of the genes was constitutive but enhanced by copper, and confirmed that the genes function both in copper resistance and production of active cytochrome c. However, two mutants in cycH were copper-sensitive and oxidase-positive, suggesting that the functions of these genes, rather than cytochrome c oxidase itself, were required for resistance to copper